The development of new cancer drugs is now based on the identification of genes that are responsible for driving malignancy and the elucidation of the signal transduction pathways that they hijack. Tremendous advances have been made in the area of small molecule kinase inhibitors to develop targeted therapies designed to interfere with critical molecules and signaling pathways driving tumor growth, for example, Imatinib® (BCR-ABL), Gefitnib® (EGFR), Erlotinib®, (EGFR) and many other agents under preclinical and clinical development. It is important to focus on the pharmacokinetic and metabolic properties as well as on target potency and molecular selectivity to optimize the effects of targeted therapies. Collin, I and Workman, Cancer Signal Transduction Therapy, 1: 3-23 (2006). In the clinical development of the kinase inhibitors, it is important to develop robust and informative biomarkers and develop assays for predicting molecular dependence and hence for identifying patients who will benefit from personalized medicine from a particular agent, and the problem of drug resistance developing.
Another challenge facing the development of molecular therapeutics is the likely need to inhibit several oncogenic targets in order to overcome cancers that are driven by several abnormalities, as well as to prevent or neutralize the development of drug resistance. In some cases resistance to a drug may be linked to increased production of molecules (e.g., cytokines, calcium channel signaling, or molecular signaling) in the tumor micro-environment that interferes with the sensitivity and efficacy of the drugs. Therefore, even the most rationally conceived drug molecule may fail because of mutational changes downstream from its intended target or metabolic features of tumors that never allow the drug to reach its target or that trigger feedback mechanism against the drug molecule.
There are several methods currently in use to overcome drug resistance. One is use of cocktails of highly targeted agents that are designed according to the molecular make-up of the specific cancer. Another approach is to use multi-targeted kinase inhibitors (for example Sorafenib®). A further strategy is to use an inhibitor of several kinases that control many oncogenic players and pathways in malignancy, for example inhibitors of histone deacetylases (HDAC) and the HSP90 molecular chaperone. Garon E. B, et al, Mol Cancer Ther., 12: 890-900 (2013); and Witt O at al, Cancer Letters 277: 8-21 (2009).
Screening and structure-based design of targeted drugs against oncogenic markers driving specific cancers are now delivering targeted drugs for preclinical and clinical studies at a rapid rate. However, there is need to study signaling pathways in normal cells to better understand the factors that cause important tumor suppressor proteins to fail as gatekeepers of normal cellular function. There is need to better understand how these tumor suppressor proteins may be modulated to prevent loss of their normal signaling in response to stress signals.
More particularly, it is important to develop drugs that can aid normal cells to integrate multiple signaling pathways or enhance their role as gate keepers to control growth and proliferation. For example, the P53 transcription factor is a major tumor suppressor protein that serves as a gatekeeper of cellular fate in multicellular organisms. P53 is activated in response to a variety of stress signals and initiates cell cycle arrest, senescence or apoptosis via pathways involving transactivation of P53 target genes. Stambolic V et al., Mol Cell: 317-325 (2001). This universal protection of genetic integrity is however impaired in many human cancers. The new paradigm is to develop agents that target the precise molecular signaling that maintains normal cell cycle, growth and proliferation.
Thus, the rational selection and development of combination treatments is extremely challenging and there is need to develop combinations of targeted drugs and or chemotherapeutic agents based on knowledge of the molecular abnormalities in particular cancers, together with the understanding of the feedback loops that apply upon blockade of a given pathway, as well as enhancing the tumor suppressor signaling pathways of p53 transcription factor and PTEN in normal and cancer cells.
Carboxyamidotriazole orotate (CTO), an orotate salt of carboxyamidotriazole (CAI) is an inhibitor of receptor-operated calcium channel-mediated calcium influx, and is shown to have anti-proliferative and anti-invasive functions in several human cancer cell lines, including human glioblastoma cells. Ge S et al. Clin Cancer Res 6: 1248-1254 (2000). By interrupting calcium mobilization as a second messenger, CAI can inhibit calcium-sensitive signal transduction pathways, including the release of arachidonic acid and its metabolites; nitric oxide release: the generation of inositol phosphates; and tyrosine phosphorylation Kohn E C et al., Cancer Res 52:3208-3212 (1992); Kohn E C et al., Proc Natl Acad Sci 92: 1307-1311 (1995); Felder C F et al. J Pharmacol Exp Therap 257: 967-971 (1990); Hupe D J et al., J Biol Chem 266: 10136-10142 (1991); Mignen O et al., J Cell Sc 118: 5615-5623 (2005); and Enfissi E et al., Cell Calcium 36: 459-467 (2004). CAI inhibits phosphorylation of cellular proteins STATS and CrkL, and induces apoptosis in imatinib mesylate-resistant chronic myeloid leukemia cells by down-regulating BCR-ABL (Alessandro et al, PLOS 7: 1-13 (2012).
In clinical studies (NCT01107522) CTO given alone was safe and tolerable without determining maximum tolerated dose, in cancer patients with different tumor types and having different genomic mutations, and CTO treatment resulted in cancers responding and demonstrating stable disease or partial response showing tumor shrinkage. Thus enormous efforts are directed to the development of molecular pharmacodynamics biomarkers of signaling outputs of CTO to design combinatorial regimens against molecular targets in different types of cancers. Current methods for assessing pathway activation in tumors involve the measurement of the drug targets, known oncogenes or known tumor suppressors. However, one pathway can be activated at multiple points so it is not feasible to assess pathway activation by evaluating just known cancer associated genes.
It is therefore important to develop the complete molecular signatures of CTO in view of its effect on signaling of multiple kinases, tyrosine kinases and calcium signal transduction pathways. The invention is related to evaluation of the response of molecular pharmacodynamics markers in response to CTO treatment in human cell or tissue samples such as anagen hairs obtained from healthy subjects or from patients, either in vivo or in vitro.
In the in vivo model, anagen hairs are obtained before dosing the patient with CTO, and at different time points after the daily dosing of a therapeutic amount of CTO is given. The patient's clinical status and blood levels of CAI are monitored during this period.
In the in vitro model the anagen hairs are obtained from an untreated subject and the hairs are treated in ex vivo cultures with different doses of CTO which represent the range of doses required for therapeutic efficacy.
In both models, RNA is extracted from the bulbs at the end of anagen hairs, cDNA is then prepared from the RNA and global transcriptional or gene expression levels are determined by microarray analysis or by quantitative PCR (qPCR). Bioinformatic analysis is then conducted to identify CTO induced gene expression changes in anagen hairs. Such a protocol can also be applied to issues other than anagen hair obtained from healthy subjects or patients.
Accordingly, the present invention describes in greater detail, uses of the plucked hair biomarker assay to study effects of CTO on mRNA and protein expression levels in vitro. Plucked scalp hair is an ideal surrogate for measuring direct response to treatment with CTO. Highly vascularized, hair follicle can respond within hours of exposure. Given this vascularization, their epithelial nature and rapid rate of proliferation, the cells in the hair bulb at the base of the plucked hair and the outer root sheath are highly relevant surrogate marker tissue for solid tumors. Highly vascularized, the hair follicle can respond to drug treatment within hours of exposure. Bioinformatic analysis was conducted to identify drug-induced changes in hairs.